Nuclear and Mitochondrial DNA from Wild-Type and Petite Yeast: Circularity, Length, and Buoyant Density

Purified mitochondrial and nuclear DNA from diploid isogenic wild-type and vegetative-petite baker's yeast were analyzed by electron microscopy and by analytical ultracentrifugation in CsCl gradients. The buoyant densities in CsCl of nuclear DNA were identical for the two strains (ρ = 1.700), but there was a difference between mitochondrial DNA from the wild type (ρ = 1.684) and the petite (ρ = 1.680). Electron microscopy revealed both circular and linear filaments for nuclear and for mitochondrial DNA of both strains. Nuclear DNA molecules included 6.5 per cent cyclic filaments principally measuring 2 μ or less in contour length, and linear filaments showing a unimodal, disperse length-distribution centered at about 2 to 3 μ, for both strains. Mitochondrial DNA for wild type varied depending upon the method used to extract and purify the molecules; showing only 7.5 per cent circular molecules from CsCl-subfractionated samples, as compared with 15 per cent circles from chloroform-extracted DNA not subjected to CsCl and up to 50 per cent circles from osmotically-lysed mitochondira, as reported in an earlier study. Modal lengths of circles occurred at about 2, 5, and 10 μ Increasing shear degradation also was evident in comparisons of the length-distribution patterns of linear molecules using the three preparative methods. Petite mitochondrial DNA contained 36-38 per cent circular molecules which measured 0.3-5.3 μ, but principally in the range of 0.3 to 2.0 μ whether from chloroform-extracted populations or from ones subfractionated in CsCl. A previous study of osmotically lysed mitochondria had shown a maximum of 8 per cent circles, which we now attribute to a failure, at that time, to detect circles measuring less than 1 μ, a substantial component encountered in the purified DNA samples in the present study. Linear filaments presented a unimodal length distribution in every case. Despite the variation in molecule populations derived from the three different preparative methods, there were consistent differences between mitochondrial DNA from wild-type and petite yeast in frequencies and size of circular molecules, as well as in length distribution patterns.